TWI764284B - Method against coronavirus infection with quinoline compound - Google Patents

Abstract

Disclosed herein is a method against coronavirus infection, which includes administering to a subject in need thereof an effective amount of 6-bromo-2-[1-(2,5-dimethylphenyl)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid or a pharmaceutically acceptable salt thereof.

Description

Methods of using quinoline compounds to fight coronavirus infection

The present invention relates to a kind of using 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid {6- bromo-2-[1-(2,5-dimethylphenyl)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid} to combat coronavirus infection.

6-Bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid is a A medicament of a compound represented by the following formula (I):

It has been reported that: 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid is effective against various RNA viruses All exhibit antiviral activity, such as enterovirus 71 (Enterovirus 71, EV71), Coxsackie viruses A6, A10, A16 and B3 (Coxsackie viruses A6, A10, A16, and B3), influenza A and B (influenza) types A and B), and human rhinovirus serotype 2 (HRV2). 6-Bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid potently inhibits RNA in the early post-infection stage - Dependent RNA polymerase 3D proteins (RNA-dependent RNA polymerase 3D proteins) and virus replication, and reduce the level of viral RNA accumulation (John TAHsu et al. (2012), Antimicrob Agents Chemother. , 56(2):647- 657; with Arul Balaji Velu et al. (2014), Antiviral Research , 112, 18-25).

Coronaviruses are a group of related RNA viruses that infect various animals including humans, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus Virus (Middle East respiratory syndrome coronavirus , MERS-CoV), human coronavirus 229E (human coronavirus 229E, HCoV-229E). Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was recently identified as a novel coronavirus. The main symptoms include respiratory symptoms such as fever above 38°C, cough, shortness of breath, and difficulty breathing. Symptoms such as loss of smell and taste, diarrhea, headache, chills, loss of appetite, general malaise, and impaired consciousness may be observed. Currently, no effective curative treatment has been established for COVID-19, but symptomatic treatment is the core.

Summary of Invention

Therefore, it is an object of the present invention to provide a method of combating coronavirus infection, which can alleviate at least one of the disadvantages of the prior art.

According to the present invention, the method comprises administering to an individual in need thereof an effective amount of 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazole-4- [methyl]-quinoline-4-carboxylic acid or a pharmaceutically acceptable salt thereof.

Detailed description of the invention

It is to be understood that if any antecedent publication is cited herein, that antecedent publication does not constitute an admission that, in Taiwan or any other country, the antecedent publication forms common general knowledge in the art one part.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and that the term "comprises" has a corresponding meaning.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Of course, the present invention is in no way limited by the methods and materials described.

The present invention provides a method of combating coronavirus infection, comprising administering to an individual in need thereof an effective amount of 6-bromo-2-[1-(2,5-xylene)-5-methanone base-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid (hereinafter referred to as "HP520-3") or a pharmaceutically acceptable salt thereof.

As used herein, the term "against coronavirus infection" or "anti-coronavirus infection" means preventing infection by a coronavirus, inhibiting the replication of a coronavirus, and/or Or treat and/or prevent infectious diseases caused by coronaviruses.

As used herein, the term "administration" or "administering" means the introduction, provision or delivery of a predetermined active ingredient into a subject by any suitable route to perform its intended function.

As used herein, the term "subject" means any animal of interest, such as humans, monkeys, cows, sheep, horses, pigs, goats, dogs, cats, mice, and rats. In certain embodiments, the individual is a human.

As used herein, the term "pharmaceutically acceptable salt" means capable of providing (directly or indirectly) a compound as described herein (ie, HP520- 3) Any salt without excessive toxicity, irritation, allergic reaction and the like. In particular, "pharmaceutically acceptable salts" may be included in those approved by regulatory agencies of the Federal or a state government, or in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly humans. those listed. The preparation of salts can be carried out by methods well known in the art.

For example, pharmaceutically acceptable salts of HP520-3 can be acid addition salts, base addition salts or metal salts, and they can be synthesized from compounds containing basic or acidic moieties by conventional chemical methods parent compound. Generally, the salts are prepared, for example, by reacting the compounds in free acid or base form with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or mixtures thereof and was produced. Examples of acid addition salts may include: mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrates, and phosphates; and organic acid addition salts such as, for example, acetate, maleate, fumarate , citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate, p-toluenesulphonate, 2-naphthalenesulphonate, and 1,2-ethanedisulphonate. Examples of base addition salts may include inorganic salts such as, for example, ammonium salts; and organic salts such as, for example, ethylenediamine salts, ethanolamine salts, N, N,N-dialkylenethanolamine salt, triethanolamine salt, choline salt, glucamine salt, and basic amino acid salt ( basic amino acids salts). Examples of metal salts may include, for example, sodium, potassium, calcium, magnesium, aluminum, and lithium salts.

According to the present invention, the coronavirus infection can be caused by a coronavirus selected from the group consisting of: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (human coronavirus 229E, HCoV-229E), and combinations thereof.

According to the present invention, HP520-3 or a pharmaceutically acceptable salt thereof can be manufactured into a compound suitable for, for example, parenteral or oral administration, using techniques well known to those skilled in the art. Pharmaceutical composition in dosage form for oral administration. Dosage forms suitable for administration include, but are not limited to, injections (such as sterile aqueous solutions or dispersions), sterile powders, tablets, troches, lozenges, capsules (capsules), dispersible powder (dispersible powder), granules (granule), solutions (solutions), suspensions (suspensions), emulsions (emulsions), syrup (syrup), elixirs (elixirs), thick syrup (slurry), and the like.

According to the present invention, the pharmaceutical composition may be administered via a parenteral route selected from the group consisting of: intraperitoneal injection injection), intrapleural injection, intramuscular injection, intravenous injection, intraarterial injection, intraarticular injection, intrasynovial injection ), intrathecal injection, intracranial injection, and sublingual administration.

According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier which is widely used in the pharmaceutical manufacturing art. For example, the pharmaceutically acceptable carrier includes one or more of the following agents: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents gelling agents, preservatives, fillers, wetting agents, lubricants, absorption delaying agents, liposomes, and the like . The selection and quantity of the aforementioned reagents fall within the scope of the professional quality and routine skills of those skilled in the art.

In accordance with the present invention, the dosage and frequency of administration of the pharmaceutical composition will vary depending on the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the individual to be treated. The daily dose of the pharmaceutical composition can be administered in a single dose or in several doses.

According to the present invention, a pharmaceutical composition comprising HP520-3 can be formulated into an external preparation suitable for application to the hands or skin [such as a hand sanitizer ( hand sanitizer) or hand washing agent]. The external preparation includes, but is not limited to: emulsion, soap, gel, ointment, cream, aerosol, spray, lotion ), serum, paste, foam, and drop.

According to the present invention, the pharmaceutical composition comprising HP520-3 is easy to apply, has low toxicity, is environmentally friendly, and is not bioaccumulative, so it can be used as an environmental disinfectant (such as a surface cleaner). cleaner), detergent (detergent), and sterilant (sterilant)].

According to the present invention, the pharmaceutical composition may further comprise remdesivir as a synergistic antiviral agent.

The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are only for illustration purposes and should not be construed as limitations on the implementation of the present invention.

Example General experimental materials: 1. Source and culture of Vero E6 cells

African green monkey kidney cells (Vero E6) were obtained from Chang Gung Medical Foundation, the Linkou Chang Gung Memorial Hospital (Taiwan). Vero E6 cells were cultured in a 10-cm Petri dish containing DMEM (Cat. No. 12000-061, Cat. No. 12000-061) supplemented with 10% FBS (Cat. No. 26140-079, Gibco). Gibco) (hereinafter referred to as "E10 medium"). Vero E6 cells were cultured in an incubator whose culture conditions were set at 37°C and 5% CO ₂ . Medium changes were performed every 2 to 3 days. Cell passage was performed when the cultured cells reached 80%-90% confluence.

2. Virus strains

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human coronavirus 229E (human coronavirus 229E, HCoV-229E) was provided by Chang Gung Memorial Hospital, Linkou Chang Gung Memorial Hospital (Taiwan).

SARS-CoV-2 and HcoV-229E were each dissolved in Dulbecco's modified Egger's medium supplemented with 2% fetal bovine serum (FBS) (Cat. No. 26140-079, Gibco). (Dulbecco's Modified Eagle's Medium, DMEM) (Cat. No. 12000-061, Gibco) (hereinafter referred to as "E2 medium") to prepare a SARS-CoV-2 solution with a viral load of 5.73×10 ⁶ pfu/mL and One HcoV-229E solution with a viral load of ^6.6 x 106 pfu/mL. The two virus solutions were kept in a freezer at -80°C for further experiments.

Example 1. Efficacy of 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid against HCoV-229E assessment experimental method:

Vero E6 cells were divided into 6 groups, including a normal control group, a pathological control group, and four experimental groups (ie, experimental groups 1 to 4). Groups of Vero E6 cells were grown at 2×10 ⁴ cells/well in each well of a 96-well culture dish containing 100 μL of E2 medium, followed by incubation in an incubator (37° C., 5% CO ₂ ) for 24 hours. Afterwards, the medium in each well was removed, and cells in experimental groups 1 to 4 were pretreated with 6-bromo-2-[1-(2,5-xylene) at concentrations of 39, 78, 156, and 312 nM, respectively. -5-Methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid (ie, HP520-3) (Cat. No. Molport-020-176-277, Molport Inc.), followed by 150 μL of the HcoV-229E solution prepared in point 2 of "General Experimental Materials" was added.

In addition, 50 μL of E2 medium was added to the cells of the pathological control group, followed by 150 μL of the HcoV-229E solution prepared in point 2 of "General Experimental Materials". The cells of the normal control group were added with 200 μL of E2 medium, and were left untreated with the HcoV-229E solution prepared in point 2 of "General Experimental Materials".

Each group was cultured in an incubator (37°C, 5% CO ₂ ) for 96 hours. The liquid from each well was removed, followed by the addition of 50 μL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide {3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyltetrazolium bromide} (MTT). After culturing for 2 hours in an incubator (37°C, 5% CO ₂ ), 150 μL of dimethyl sulfoxide (DMSO) was added to each of the obtained cell cultures, followed by an ELISA reader ( ELISA reader) The resulting mixture was subjected to measurement of absorbance at a wavelength of 590 nm.

The cell viability ratio (%) was calculated by the following formula (I): A=(B/C)×100 (I)

Where: A = cell viability ratio (%)

B = OD ₅₉₀ value of each group

C = OD ₅₉₀ value of normal control group

In addition, the 50% effective concentration ( _EC50 ) was derived from the dose plotted by the active ingredient concentration (n=3) that reduced the absorbance of the treated cells by 50% (compared to the pathological control group) - The linear part of the response curve is determined. Experimental data are expressed as mean ± SD [standard deviation].

result:

Figure 1 shows the cell viability ratios for each group. As can be seen from Figure 1, the cell viability ratios measured in experimental groups 1 to 4 were significantly higher than those measured in the pathological control group, and HP520-3 exhibited dose-related antiviral effects. In addition, the _EC50 value of HP520-3 was 140.7 ± 15.45 nM.

Summarizing the above test results, it is clear that HP520-3 is effective against HcoV-229E infection.

Example 2. Evaluation of the efficacy of HP520-3 against SARS-CoV-2 A. Viral plaque reduction assay:

Vero E6 cells were divided into 3 groups, including a pathological control group and two experimental groups (ie, experimental groups 1 to 2). Groups of Vero E6 cells were grown at 4× ^{10 5} cells/well in each well of a 24-well dish containing 0.5 mL of E2 medium, followed by incubation in an incubator (37° C., 5% CO ₂ ) for 24 hours. Afterwards, the medium in each well was removed, and cells of each group were infected with SARS-CoV-2 at a multiplicity of infection (moi) of 0.01. After 1 hour of incubation in the incubator (37°C, 5% CO ₂ ), the liquid in each well was removed and the groups were washed with phosphate-buffered saline (PBS) after SARS- CoV-2-infected Vero E6 cells twice.

Thereafter, the SARS-CoV-2-infected Vero E6 cells of experimental group 1 were covered with E2 medium containing 1.4% methyl cellulose and 1 μM HP520-3, and the SARS-CoV-2 infected Vero E6 cells of experimental group 2 were overlaid with E2 medium containing 1.4% methyl cellulose and 1 μM HP520-3. -2-Infected Vero E6 cells were overlaid with E2 medium containing 1.4% methylcellulose and 10 μM HP520-3, and SARS-CoV-2-infected Vero E6 cells of the pathological control group were overlaid with 1.4% formazan cellulose-based E2 medium.

After culturing in an incubator (37°C, 5% _CO2 ) for 72 hours, cells of each group were fixed with 0.5 mL of a 4% paraformaldehyde solution for 1 hour at room temperature. Afterwards, the fixed cells in each well were stained with 1% crystal violet for 20 minutes. After washing the stained cells with water, the distribution of viral plaques in each well was analyzed by visual inspection, and the number of viral plaques in each group was counted.

result:

Referring to Figure 2 to Figure 3, the number of viral plaques measured in experimental groups 1 to 2 were lower than those measured in the pathological control group, indicating that HP520-3 can inhibit SARS-CoV-2-infected host cells effective in reducing viral replication.

B. Quantitative determination of viral gene expression:

Vero E6 cells were divided into 4 groups, including a pathological control group and three experimental groups (ie, experimental groups 1-3). Groups of Vero E6 cells were grown at 4× ^{10 5} cells/well in each well of a 24-well dish containing 100 μL of E10 medium, followed by incubation in an incubator (37° C., 5% CO ₂ ) for 24 hours. After that, the medium in each well was removed, and the cells of experimental groups 1-3 were pretreated with HP520-3 at a concentration of 0.3125 μM, 1.25 μM, and 5 μM, respectively, and then treated with SARS-CoV at a multiple of infection (moi) of 0.01. -2.

In addition, cells from the pathological control group were treated with SARS-CoV-2 at a multiple of infection (m.o.i) of 0.01, but not HP520-3.

Each group was incubated in an incubator (37°C, 5% CO ₂ ) for 1 hour. The liquid from each well was collected and total RNA was extracted using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) according to the manufacturer's instructions.

Thereafter, the obtained RNA (1 μg) of each group was used as a template and subjected to reverse transcription polymerase chain reaction using M-MLV reverse transcriptase (Invitrogen, USA). , RT-PCR) to synthesize cDNA. The cDNA thus obtained was used as a DNA template, diluted 100-fold with deionized distilled water, and subjected to reverse transcription quantitative polymerase chain reaction (RT-qPCR) [which was performed using TaqMan ^™ qRT]. - PCR panels and reaction conditions shown in Table 1 were performed on a StepOnePlus real-time PCR system (Applied Biosystems)] to determine changes in envelope (Envelope, E) gene expression. Gene expression data were normalized using the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) gene as an endogenous control in quantitative analysis of RT-qPCR.

SARS-CoV-2-E gene-specific primer pair and GAPDH gene-specific primer pair were purchased from TaqMan (Thermo Fisher Scientific). Details of the aforementioned primer pairs are summarized in Table 2.

Table 2

To quantify changes in gene expression, the threshold cycle method [threshold cycle( ΔCT ₎ method] was used to calculate relative fold changes normalized to the GAPDH gene.

result:

Figure 4 shows the relative RNA expression levels of the E gene of SARS-CoV-2 in Vero E6 cells infected and pretreated with HP520-3. It can be seen from Figure 4 that the RNA expression levels of experimental groups 1 to 3 are significantly lower than those of the pathological control group, indicating that HP520-3 can reduce viral gene expression in host cells, thereby inhibiting viral replication.

All patents and documents cited in this specification are incorporated by reference in their entirety. In the event of conflict, the detailed description of the case (including definitions) will prevail.

Although the present invention has been described with reference to specific examples which are considered to be exemplary, it is to be understood that this disclosure is not limited to the specific examples disclosed, but is intended to cover the spirit and scope included in the broadest interpretation. Various configurations, to include all such modifications and equivalent configurations.

The above and other objects, features and advantages of the present invention will become apparent upon reference to the following detailed description and illustrative examples and accompanying drawings, wherein: Figure 2 shows the distribution of viral plaques in each group described in item A in Example 2 below; Figure 3 shows the number of viral plaques in each of the groups described in Item A of Example 2 below; and Figure 4 shows the relative RNA expression levels in each of the groups described in Item B of Example 2 below.

Claims (4)

A kind of 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid {6-bromo-2-[1 -(2,5-dimethylphenyl)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid} or a pharmaceutically acceptable salt thereof is supplied for the preparation of a compound for use against coronavirus infection Use of pharmaceuticals. The use of claim 1, wherein the coronavirus infection is caused by a coronavirus selected from the group consisting of: severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome Acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), Middle East respiratory syndrome coronavirus (Middle East respiratory syndrome coronavirus , MERS-CoV), human coronavirus 229E (human coronavirus 229E, HCoV-229E ), and their combinations. The use of claim 1, wherein the 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid or The pharmaceutically acceptable salts are in a dosage form for oral administration. The use of claim 1, wherein the 6-bromo-2-[1-(2,5-xylene)-5-methyl-1H-pyrazol-4-yl]-quinoline-4-carboxylic acid or The pharmaceutically acceptable salts thereof are in a dosage form for parenteral administration.

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